Flowering is an age (development)-dependent response which occurs following a period of vegetative growth in which the plant acquires the competence to flower. The timing of flowering is highly regulated to ensure effective pollination and that seed maturation occurs under favorable environmental conditions.
The earliest stage of flowering, floral evocation, is characterized by the normally irrevocable commitment of shoot apical meristem (SAM) to form an inflorescence or floral meristem. Floral evocation occurs prior to the appearance of detectable morphological or functional changes in the SAM. Subsequently, the floral primordium differentiates and forms floral organs, the flower matures and opens.
Environmental inputs are also involved in the onset of floral evocation. In certain species of plants, floral evocation will not occur in the absence of an appropriate environmental stimulus (e.g., daylength (photoperiodism), cold temperatures (vernalization) and/or other environmental stresses such as drought), while in other species, floral evocation will occur at a time that is predetermined genetically for that species but is susceptible to modulation by the environment. Genetic studies in Arabidopsis have provided information about the organization and integration of flower signaling pathways in this model system in which there exists three flower signaling pathways: an age-dependent, daylength-independent pathway which operates in the absence of promoting signals; an autonomous pathway which suppresses the vegetative-to-flowering transition; and a photoperiodic pathway, which in this species of plant, is triggered when the photoperiod exceeds a critical length (reviewed by Devlin and Kay, Science 288: 1600 (2000)).
Flowering control in Arabidosis is normally described as controlled by 4 pathways:
1) Autonomous promotion
2) vernalization promotion
3) gibberellic acid promotion
4) photoperiod promotion
Simpson, Gendall and Dean, Ann. Rev. Cell Dev. Biol. 99: 519–550, 1999.
An understanding of the flower control mechanisms that operate in agronomically and horticulturally important plants is a prerequisite for being able to control the occurrence, timing and extent of flower and seed production. The ability to override, or change, the genetic programs of flowering plants, to accelerate flowering or to permit flowering under environmental conditions different from those of normal, without altering the fertility of the plants or their desirable traits would be of tremendous importance in agriculture and horticulture. The acceleration of flowering is useful in plant breeding to increase the efficiency and speed of selection of improved traits, and in the ornamental flower industry. The ability to delay flowering is important in certain crop plants in which flowering overlaps with growth of harvested vegetation, such as onions, bulbs, lettuce, cabbage and pastures.
One of the most useful paradigms for studying the control of flowering is the photoperiodic response. In many plant species, mature leaves serve as photoreceptors for photoperiodic induction leading to floral initiation within the vegetative meristem. Although the identity of the transmitted flowering stimulus is presently unknown, grafting and defoliation experiments have provided irrefutable evidence that a flowering stimulus is produced in the leaves of plants exposed to a favorable photoperiod. Upon stimulation, a phloem-mobile agent, termed florigen, appears to be released from leaf cells for translocation and delivery into the terminal phloem cells within the apex of the plant. Relay of the florigenic signal from the phloem to the founder cells within the SAM then gives rise to the activation of a new developmental program that generates floral meristems and, eventually, flowers. Available evidence suggests that flower development in certain species of plants may be controlled by both positive and negative regulatory factors, the latter at times being produced in response to an unfavorable photoperiodic stimulus. See, e.g., Gibby and Salisbury, Plant Physiol. 47:784–789 (1971); Lang et al., Proc. Natl. Acad. Sci. USA 74:2412–2416 (1977).
Significant progress has been made in identifying structural and regulatory genes involved in pathways of flower development and in understanding the hierarchical controls that integrate multiple pathways. See, e.g., Levy and Dean, Plant Cell 10:1973–1989 (1998); Simpson et al., Annu. Rev. Cell Dev. Biol. 99:519–50 (1999). Multiple environmental and endogenous signals are integrated by floral integrator genes, which participate in the upregulation of floral meristem identity genes which is required for flowering.
Genetic analysis in Arabidopsis has revealed many of the genes involved in photoperiod-mediated flowering. In this pathway, the timing of the floral transition is coordinated with day-length, as sensed by the circadian oscillator. The transition is accelerated in response to long days and retarded in response to short days. CO (CONSTANS), a transcription factor, plays an important regulatory role in this process. The level of CO mRNA is circadian clock-regulated. Under long day length conditions (LD), CO protein accumulates late in the day and directly activates transcription of FT (FLOWERING LOCUS T), a floral integrator gene. The level of CO-activated FT expression in Arabidopsis is tightly regulated by TFL2 (TERMINAL FLOWER 2), a gene that is coexpressed with CO and FT in leaf vascular tissue (Takada and Goto, The Plant Cell 15, 2856–2865 (2003)).
FT encodes a RAF-kinase-inhibitor-like protein that promotes flowering. The Arabidopsis FT polynucleotide and polypeptide sequences and methods of use for accelerating or delaying flowering are disclosed in U.S. Pat. Nos. 6,225,530 and 6,713,663 respectively.
CO also activates LEAFY (LFY), a floral meristem identity gene. FT, together with LFY, promotes flowering. The loss of FT delays flowering, whereas overexpression of FT accelerates flowering by a CO- and photoperiod-independent process.
FT belongs to a gene family which is conserved in plants, mammals and fungi. Two known plant family members are CEN (Antirrhinum majus) and TFL1 (Arabidopsis thaliana). TFL1 is required for establishing and maintaining the inflorescence meristem. TFL1 and CEN are orthologous genes according to phylogenetic tree analysis of the protein sequences (see U.S. Pat. No. 6,225,530). FT, acting downstream of CO, promotes flowering, whereas TERMINAL FLOWER1 (TFL1) represses flowering.
Flowering promotion by FT requires FD, a bZIP transcription factor-encoding gene. FD is preferentially expressed in the shoot apex and activates transcription of the floral meristem identity genes AP1 and CAL. The overexpression of FD in seedlings upregulates AP1 expression only when FT is present. The interaction of FD with FT may require phosphorylation of the C-terminus of FD. (Daimon et al., Abstract T01–033, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
TFL1 interacts with FDP, a bZIP transcription factor which is closely related to FD. The interaction of FDP and TFL1 may be required to prevent inappropriate activation of flowering by FD and FT in the SAM (Wigge et al., Abstract T01–070, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
Reportedly, both TFL1 and FT protein move from cell to cell in the SAM. (Goto and Nakayama, Abstract T01–053, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
Unpublished studies from our laboratory have shown that ectopic expression of the Arabidopsis FT gene in the vasculature of Cucurbita moschata, grown under non-flowering photoperiod conditions, promotes flowering in the meristem. We used a viral vector system based on Zucchini yellow mosaic potyvirus (ZYMV) to express FT (ZYMV-FT) in C. moschata PI441726. In contrast to control plants inoculated with ZYMV-GFP, plants inoculated with ZYMV-FT formed floral buds and flowered within 23–35 days. These results were obtained in replicate independent experiments with four to six plants per experiment. Representative results are shown below in FIGS. 147 and 148. Our results show that expression of FT in the vasculature sends a signal to the meristem to initiate flowering. Our results are consistent with the possibility that FT or a peptide fragment of FT acts as the signal, as potyviruses are normally excluded from the meristem of plants (Jones et al., EMBO J. 17:6385–6393, 1998). However, as discussed in the Detailed Description below, these results are also consistent with the possibility that FT participates in a signaling complex with other interacting molecules and that one of these interacting molecules, or a different gene altogether, comprises the long-distance florigenic signal
Coupland and colleagues expressed CO::GUS promoter-reporter gene constructs in Arabidopsis and found expression of the reporter gene in both vascular tissue and the SAM. Mis-expression of CO from phloem-specific promoters, but not meristem-specific promoters, induced early flowering and complemented a late-flowering constans mutation. CO activates flowering through both FT-dependent and FT-independent processes (Laurent et al., Abstract T01–048, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
Micrografting experiments in Arabidopsis have shown that certain flowering time mutants can be rescued by long-distance signaling, and suggest that the expression of FT in the apex and leaf may be required (Turnbull and Justin, Abstract T01–099, 15th International Conference on Arabidopsis, Berlin, Germany, Jul. 11–14, 2004).
The CEN, TFL1, FT and SP (SELF-PRUNING) genes are a closely related set of plant genes which are implicated as regulators of timing of switching of meristems from vegetative to reproductive growth (all except FT delay flowering) (Bradley et al., Science 275:80–83 (1997); Bradley et al., Nature 379:791–797 (1996); Pneuli et al., Development 125:1979–1989 (1998); Pneuli et al., The Plant Cell, 13:2687–2702 (2001). This gene family, recently named CETS (Pneuli et al., Ibid)), is comprised of plant homologs of the mammalian phosphatidylethanolamine binding proteins (PEBPs), which include serine protease inhibitors, Raf-1 kinase inhibitor (RKIP), and precursor for hippocampal neurostimulatory peptide (HCNP). Six CETS members have been identified in Arabidopsis and six in tomato.
Two-hybrid screens and in vitro binding assays have revealed the presence of SP-interacting proteins (SIPS) in tomato (Pneuli et al., 2001). These SIPs include: SPAK, a novel plant serine-threonine NIMA-like kinase; several isoforms of the 14-3-3 family of adapter proteins; and SPGB, a putative bZIP G-box binding transcription factor C-terminal sequence similarity to GBF4.
SIPs may function as components of an SP-dependent signaling network in tomato plants. SIPs form specific associations with one another and show overlapping spatio-temporal expression patterns in apical meristem, leaf and stem vasculature, floral primordial of the primary inflorescence, stamens and carpels in developing floral bud and vegetative meristem of the first sympodial segment (Pneuli et al., 2001, Ibid). Several of the binding interactions are phosphorylation-dependent. For example, the binding of SP to SPAK and the binding of SIP4 and SPAK to 14-3-3/74 requires phosphorylation of SPAK at Serine 406. Both CEN and TFL1 bind SPAK, 14-3-3/74 and SPGB proteins but not SIP4 (a novel 10 kDa protein). FT showed the same binding pattern as TFL1.
The biological functions of SP/14-3-3/SPAK interactions in tomato plants are not yet understood. Nevertheless, the finding that CETS proteins of Arabidopsis and Antirrhinum form specific complexes with tomato SIPs suggests that protein-protein interactions may be necessary for CETS protein function in plants with widely differing shoot and flowering architecture.
PCT Application No. WO 96/34088 discloses the isolation of the Id gene, which is thought to be important in regulating the transition to flowering in maize. The Id gene encodes a zinc-finger protein that is apparently transcribed in young leaves but not in the shoot apical meristem. The mechanism by which this gene produces its effects at the SAM has not yet been elucidated. Mutated forms of the gene are proposed for use in accelerating or delaying floral induction in a plant. PCT Application No. WO 97/25433 describes chimeric vectors comprising FPF (flowering promoting factor) genes from mustard, and homologous genes from other plants, and uses thereof for inducing early flowering or inhibiting flowering in various crop plants.
Progress has also been made in understanding how plants transport materials from cell-to-cell and systemically throughout the plant. These studies have provided evidence for a systemic communication network that comprises a phloem translocation system which is capable of transporting not only small phytohormones and nutrients but also macromolecules (such as peptides, proteins and nucleic acids) between spatially distant tissues and organs of the plant. The identification of specific transcripts and signaling molecules in phloem sap suggests that this communication network is involved in the coordination of growth and development, and may participate in systemic acquired resistance to pathogens (Narvaez-Vasquez et al., Planta 195:593–600 (1995), systemic gene silencing (Jorgensen et al., Science 279:1486–1487 (1988), review), biomass distribution, regulation of carbon metabolism and control of plant size (Lucas, PCT applications WO 97/06669 and WO 97/20470), and floral development (Ruiz-Medrano et al., Development 126:4405–4419 (1999) and references cited therein; Baulcombe, PCT application WO 99/15682). A systemic small RNA binding protein which may function in sRNA signaling has been isolated from several plant species and is disclosed in U.S. Application Ser. No. 60/487,358.
The broad outlines of florigen signaling have emerged from these and prior studies, but the signal(s) that initiate floral evocation and the manner in which the long-distance florigen signaling pathway is regulated are presently unknown. These objectives are addressed in the present application.